Otitis media (OM) or middle ear infection remains an important public health problem in the US. OM is the top reason for clinical consult and antibiotic use in US children, with annual cost >$5 billion. Worldwide, incidence and prevalence of OM are high in sub-Saharan Africa and Asia, and in marginalized communities such as indigenous populations. Strong evidence exists for genetic susceptibility to OM, but no rare OM susceptibility variants were previously reported. This study aims to: (1) Identify rare OM susceptibility variants within families and in probands; and (2) Identify differences in the middle ear microbiome due to OM susceptibility variants. In this study OM is treated as a disease spectrum such that all individuals with acute, chronic, effusive or healed OM are considered affected. This strategy has allowed initial success in variant and microbial profile identification and will increase power to detect significant associations. Cohorts to be studied for OM susceptibility variants include: (1) a large indigenous Filipino pedigree with high OM prevalence and evidence for intra-familial locus heterogeneity for OM susceptibility variants; (2) a case-control cohort of otitis- prone and non-otitis-prone childre who were followed from birth; (3) 143 families with chronic or recurrent OM; and (4) at least 600 trios that have a proband who will undergo surgery for chronic or recurrent OM. For the indigenous Filipino population and surgical patients from the Philippines, microbial gene profiling will be performed as well, and overlap with OM susceptibility variant identification studies will allow study of microbial profiles based on variant carriage. Even though each cohort is genetically distinct they are also admixed, thus allowing for replication of rare variants across cohorts. Although standard methods on linkage analysis, DNA and microbial sequencing will be used, new techniques in rare variant and microbial analysis will be applied, including the rare variant-transmission disequilibrium test for NGS data using OM trios and in-house pipelines for rare variant identification using NGS data and microbial gene profiling. Novel OM susceptibility genes will be followed up by mouse middle ear localization of protein products and creation of mutant constructs for functional analysis. Additionally screening for novel variants within previously identified OM susceptibility genes will be performed for newly recruited trios and families. For microbiome studies, middle ear samples will include swabs on discharge and mucosa, as well as mucosal tissue and cholesteatoma. Microbial diversity and relative abundance will be studied based on OM chronicity and carriage of OM susceptibility variants. Identification of rare variants that confer OM susceptibility and cause changes in the middle ear microbiome can illuminate pathways that are involved in OM pathophysiology, which in turn can be targeted for OM prevention and treatment, for example, by new drug development, antibiotic selection and vaccine prioritization guided by genotype and/or microbial profile.